| Objective To clone and construct the recombinant adeno-associated virus vector containing the full length of cDNA coding mouse membrane form KL. The ovariectomized rats (OVXs) were transfected with rAAV.mKL, to examine the effect of KL Protein on the bone metabolism and bone microstructure, to study the regulation mechanisms of KL on bone metabolism of osteoporosis.Methods (1) Total mRNAs were extracted from mouse kidney tissue, then the full length of KL cDNAs were obtained by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pAAV-HnGFP recombinant plasmids that had been constructed. The recombinant plamids were transfected into COS-7eukaryotic expression cells, and the expression in COS-7cells were detected by fluorescent microscopy, RT-PCR and Immunofluorescence and ELISA.The pAAV-mKL was then transfected into AAV-293with pHelper and pAAV.RC to produce rAAV.mKL.(2) Transfect OVXs with rAAV.mKL, to examine the effect of KL Protein on the bone metabolism and bone microstructure.Female SD rats were randomly divided into sham operation group (S group) and model group.Model was successfully constructed with ovariectomy after12weeks, they were randomly divided into model group (O group),17β-estradiol (E group), KL gene group (KO group), empty vector group (GO group), all were sacrificed after12weeks. KL, osteocalcin (BGP) and estradiol expression and production were measureed by ELISA.Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry in the femurs and tibia. The fluorescent expression of renal KL was observed by Cryo-sectioning Technique.The Runx2and MMP-13mRNA expression of bone tissue were detected by RT-PCR.Expression of KL protein in kidney and Runx2,MMP-13protein in bone were detected by immunohistochemistry. Bone morphological changes of the different groups were observed by HE staining and TEM examination.Results (1) The full length of cDNA was obtained by RT-PCR, the pAAV.mKL was constructed successfully, the expression in COS-7cells was detected.The rAAV.mKL was produced after pAAV-mKL transfected into AAV-293with pHelper and pAAV.RC. The mKL cDNA was amplified from rAAV.mKL DNA.(2) The rAAV.mKL delivery may attenuate the progression of osteoporosis and improved the destruction of bone microstructure in OVXs. KL, osteocalcin (BGP) and estradiol expression and production were suppressed in O group,which were reverted in KO group by KL delivery increased plasma levels of KL and BGP.BMD of KO and E groups were significantly higher than those in the O and GO groups(P<0.05). Specific expression of mouse KL was seen only in KO group.Renal KL protein in KO group were increased obviously by immunohistochemistry.Compared to O group,the expression of Runx2mRNA increased greatly,but the expression of MMP-13mRNA decreased in bone tissue of KO group (P<0.05). Immunohistochemistry analysis showed that the average absorbance of Runx2in KO group,it was a significant higher than that of O group(P<0.05). The average absorbance of MMP-13in KO group, it showed a significantly lower than those in O group (P<0.05).The trabecular bones in KO,E,S groups tightly packed, interconnected to form a network,had relatively complete histologic structure. It had little or no myelin figure in bone cells to be found by the TEM examination in KO and S group.Conclusion The recombinant adeno-associated virus vector containing the full length of cDNA coding mouse membrane form KL was constructed successfully,it could increase the expression of KL in COS-7cell.The transfection of rAAV.mKL may attenuate the progression of osteoporosis and improved the destruction of bone micro structure in OVXs. KL gene may play a role in the progression of osteoporosis. |
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